This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objective of this project was to evaluate Mycobacterium tuberculosis Alanine Proline Rich Antigen (Apa) and three other recombinant mycobacterial proteins for the immunoreactivity and development of a new generation prophylactic mucosal subunit vaccine against tuberculosis (TB) in the experimental mouse model. TB is primarily a disease of the lungs, and we hypothesized that the best way to protect against infection and disease is to vaccinate via the respiratory route to develop immunity in the lungs and associated respiratory tract. Here, we characterized in detail the magnitude, quality and epitope specificity of immune responses against the native and recombinant vaccine candidate Apa in BCG- vaccinated human subjects in vitro and in BCG vaccinated mice in vivo. We characterized the immune responses elicited by these two forms of proteins following immunization of mice by intranasal and subcutaneous route. In addition, we tested the ability of an Apa based vaccine to boost BCG induced immune responses in mice model to develop a booster vaccine which can be administered in BCG vaccinated adolescents or adults. These studies demonstrated that the Apa based mucosal subunit vaccine can serve as a strong booster immunization for BCG vaccine and is expected to contribute to the development of more efficacious prophylactic mucosal subunit vaccine or a booster vaccine to existing BCG vaccine against tuberculosis.